Anti-Tbx6 antibody (ab30946)

This is China Jingmei Biotech Ltd. A customer used ab30946 to do western-blot. She added 34-40ug protein per lane for 10% SDS-polyacrylamide gels ,and she used any standard western blot protocol for immunodetection of Tbx6 protein. Unfortunately there is not specific-band,no visible background bands either. She has repeated the experiment 3 times, she cannot get the specific band until raising primary antibody concentration to a very high level.

Below is the questionnaire:

1. Order details: · Batch number: Lot. #194791 · Abcam order or Purchase order number: Cat.# ab30946 · Antibody storage conditions (temperature/reconstitution etc) At -20 °C

2. Please describe the problem (high background, wrong band size, more bands, no band etc). No band!!! 3. On what material are you testing the antibody in WB? Species: mouse. Cell extract or Nuclear extract: whole cell extract. Purified protein or Recombinant protein: over-expression and endogenous protein in cell lines.

3. The lysate · How much protein was loaded: 34-40 ug · What lysis buffer was used: RIPA lysis buffer · What protease inhibitors were used: Cocktail · What loading buffer was used: 5 X SDS loading buffer available at Takara Bio. Catalog Online (http://www.takara.com.cn) · Did you heat the samples: temperature and time: 95-100 °C 10min

4. Electrophoresis/Gel conditions/ Transfer conditions · Reducing or non reducing gel: Denature gel · Gel percentage : 10 % PAGE · Transfer conditions: 100V 2h at 4°C 5. Blocking conditions · Buffer: TBST · Blocking agent: milk, BSA, serum, what percentage: 5 % milk · Incubation time: 2 hours · Incubation temperature: room temperature

6. Primary antibody ·Specification (in which species was it raised against): Rabbit polyclonal antibody. At what dilution(s) have you tested this antibody: use 1:500 for the first time and 1:100 later at a concentration of 2-10 ug/ml. What dilution buffer was used: 5% milk in TBST. Incubation time: 4 °C overnight. Incubation temperature: room temperature. What washing steps were done: TBST 3-5 times at every 5 min

7. Secondary Antibody · Specification (in which species was it raised against)? Goat anti rabbit IgG-HRP. · At what dilution(s) have you tested this antibody: At 1:10000 · Incubation time: 1.5-2 hours at room temperature. · Wash steps: TBST 3-5 times at every 5 min. · Do you know whether the problems you are experiencing come from the secondary? Yes, we dotted the secondary antibody on the PVDF membrane, it performed successfully. It also works well in western blot using other primary antibody.

8. Detection method ECl, ECl+, other detection method: ECL detection system: SuperSignal West Pico Chemiluminescent Substrate kit (Pierce, product # 34080);

9. Background bands Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control): Sorry, we do not try, since it has few background bands. Is the blocking step sufficient? Yes, 2h at room temperature is sufficient. Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) 0.1 % Tween-20 in TBS is sufficient. At what size are the bands migrating? Could they be degradation products of your target? No visible background bands. Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient) The western-blot picture is kept in the accessory.

10. Optimization attempt How many times have you tried the Western? 3 times Do you obtain the same results every time e.g. are background bands always in the same place? No band each time. What steps have you altered? At higher concentration of primary antibod 11. Did you apply positive and negative controls along with the samples? Please specify. We use human 293T cell lysate transfected with mouse Tbx6 expression construct as a positive control, and 293T cell lysate transfected with empty vector as another negative control.

Dear sir/madam, based upon this result, we felt the antibody has a much lower titer than stated. So she asks refund. So would you please issue a check note for her? Thank you very much

ANSWER:

Thank you for contacting us on behalf of a customer.

I'm sorry to hear your customer is not having any signal with ab30946. This antibody has not been tested in mouse tissue, only in human samples and I would like to recommend to run a positive control of Jurkat whole cell lysate to make sure the problem is not due to the fact that the antibody does not recognise mouse protein. Furthermore, the customer says " We use human 293T cell lysate transfected with mouse Tbx6 expression construct as a positive control, and 293T cell lysate transfected with empty vector as another negative control"; the problem may therefore also be that levels of Tbx6 are very low and therefore not detectable. Running the positive control will also address this possibility.

As the antibody has not been tested in mouse I cannot offer the customer a credit note/refund, thank you for explaining our Abpromise policy to the customer. If she still has not results with the Jurkat lysate I would be happy to offer her a refund,